Directed evolution of an enantioselective lipase

被引:191
作者
Liebeton, K
Zonta, A
Schimossek, K
Nardini, M
Lang, D
Dijkstra, BW
Reetz, MT [1 ]
Jaeger, KE
机构
[1] Max Planck Inst Kohlenforsch, D-45470 Mulheim, Germany
[2] Ruhr Univ Bochum, Lehrstuhl Biol Mikrooganismen, D-44780 Bochum, Germany
[3] Univ Groningen, Biophys Chem Lab, NL-9747 AG Groningen, Netherlands
来源
CHEMISTRY & BIOLOGY | 2000年 / 7卷 / 09期
关键词
directed evolution; enantioselectivity; random mutagenesis; saturation mutagenesis; Pseudomonas aeruginosa lipase;
D O I
10.1016/S1074-5521(00)00015-6
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity. Results: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions. Conclusions: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening UP a large area of novel applications in biotechnology.
引用
收藏
页码:709 / 718
页数:10
相关论文
共 56 条
[1]  
Ahuja S., 1997, Chiral Separations Applications and technology, V1st
[2]   Directed evolution of biocatalysts [J].
Arnold, FH ;
Volkov, AA .
CURRENT OPINION IN CHEMICAL BIOLOGY, 1999, 3 (01) :54-59
[3]   IMPROVED METHOD FOR PCR-MEDIATED SITE-DIRECTED MUTAGENESIS [J].
BARETTINO, D ;
FEIGENBUTZ, M ;
VALCARCEL, R ;
STUNNENBERG, HG .
NUCLEIC ACIDS RESEARCH, 1994, 22 (03) :541-542
[4]   Crystallography & NMR system:: A new software suite for macromolecular structure determination [J].
Brunger, AT ;
Adams, PD ;
Clore, GM ;
DeLano, WL ;
Gros, P ;
Grosse-Kunstleve, RW ;
Jiang, JS ;
Kuszewski, J ;
Nilges, M ;
Pannu, NS ;
Read, RJ ;
Rice, LM ;
Simonson, T ;
Warren, GL .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1998, 54 :905-921
[5]   Directed evolution of a fungal peroxidase [J].
Cherry, JR ;
Lamsa, MH ;
Schneider, P ;
Vind, J ;
Svendsen, A ;
Jones, A ;
Pedersen, AH .
NATURE BIOTECHNOLOGY, 1999, 17 (04) :379-384
[6]  
Collins A.N., 1992, CHIRALITY IND COMMER
[7]  
COLLINS AN, 1997, CHIRALITY IND, V2
[8]  
Eckert K A, 1991, PCR Methods Appl, V1, P17
[9]  
Faber K, 1997, BIOTRANSFORMATIONS O, DOI 10.1007/978-3-319-61590-5
[10]  
Guo JH, 1999, ANGEW CHEM INT EDIT, V38, P1755, DOI 10.1002/(SICI)1521-3773(19990614)38:12<1755::AID-ANIE1755>3.0.CO